Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

Apolipophorin-III in the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae).

The level of apolipophorin-III reached a maximum in the haemolymph of Galleria mellonella at the end of the feeding phase of the seventh larval instar and declined to a plateau value in the pupal and the adult stages. Apolipophorin-III was detected immunologically in fat body tissue, haemocyte lysates, and plasma. In its native state, apolipophorin-III may be associated with another protein with an apparent molecular mass of 77 kDa, possibly apolipophorin-II. Injections of octopamine did not cause lipid loading of high density lipophorin.     

Lipophorin uptake by the larval fat body and adult ovary in the wax moth Galleria mellonella

Lipophorin (Lp) acts in the circulation of insects to selectively deliver lipids to target tissues. In the present study, we wanted to show that Lp is taken up into larval fat body cells and the adult ovary in Galleria mellonella. Larval fat body and adult ovary tissues were incubated at room temperature for 30 min with fluorescein isothiocyanate (FITC)‐labeled Lp. Fluorescence microscopy and sodium dodecylsulfate (SDS)–polyacrylamide gel electrophoresis (PAGE) revealed that fat body and ovary tissues internalize fluorescence‐labeled Lp. The results suggest that both lipids and proteins are taken up by fat body cells and the ovary and also that large amounts of proteins and lipids taken up can serve as building blocks and as a source of energy. Immunological relationships with other insects were investigated using western blotting. The data showed that the Lp of Galleria mellonella is related to that of Hyphantria cunea.     

Elastase B of Pseudomonas aeruginosa stimulates the humoral immune response in the greater wax moth, Galleria mellonella

The role of Pseudomonas aeruginosa elastase B in activation of the humoral immune response in Galleria mellonella larvae was investigated. The results of our study showed that elastase B injected at a sublethal concentration was responsible for eliciting the humoral immune response in G. mellonella larvae. The insects exhibited increased antibacterial activity, namely, we observed appearance of antimicrobial peptides and a higher level of lysozyme in cell-free hemolymph. Elastase B seems to be a more potent elicitor than thermolysin because similar maximal antibacterial activity levels were observed at a 5-fold lower concentration. We also demonstrated that there were differences in the kinetics of induction of antimicrobial activity between thermolysin and elastase B. The maximum level was observed 18 h post challenge of thermolysin and 38 h after injection of elastase B. It was also shown that, 24 h after elastase injection, the relative levels of apoLp-III in the hemolymph significantly increased in comparison with control G. mellonella larvae. The activation of immune responses in metalloproteinase-challenged larvae involved synthesis of metalloproteinase inhibitors which increased the survival rates of insects both against the lethal dose of thermolysin as well as against viable pathogenic bacterial cells of P. aeruginosa.     

Purification and characterization of a male-specific protein in the hemolymph of the wax moth,Galleria mellonella L.

A male-specific protein (MSP) present only in males was identified from the hemolymph of the wax moth, Galleria mellonella L., by polyacrylamide gel electrophoresis (PAGE) and purified by anion-exchange chromatography. MSP has a native molecular mass of 55 kDa and consists of two 27-kDa subunits. An isoelectric point of MSP was measured to be approximately 5.8. MSP is a glycoprotein that contains 1.7% carbohydrate. The compositional analysis of carbohydrate component indicated a predominance of fructose and glucose. MSP also contains large amounts of asparagine, aspartic acid, glutamine, glutamic acid, and lysine but small amounts of tyrosine, methionine, and tryptophan. Western blot analysis of the hemolymph of each developmental stage indicated that MSP is present in the hemolymph of 8-day-old pupa and adult. Also, results from Western blotting indicated that MSP is not present in the tissues of larvae and of female adults but appears in the fat body of male pupae and adult and testis of adult. The fat body and testis of male pupae and adult were cultured in vitro to trace the place and time of MSP synthesis. The fat body began to synthesize MSP in late pupae and showed active synthesis during the adult stage. The distribution of MSP in the testis was observed by electron microscopic immunogold labeling, using the antibody against MSP. MSP is present between the germinal cysts and is taken up through the basal surface of the seminiferous tubular epithelium. Arch. Insect Biochem. Physiol. 37:257–268, 1998. © 1998 Wiley-Liss, Inc.     

Immunity of the greater wax moth Galleria mellonella

Investigation of insect immune mechanisms provides important information concerning innate immunity, which in many aspects is conserved in animals. This is one of the reasons why insects serve as model organisms to study virulence mechanisms of human pathogens. From the evolutionary point of view, we also learn a lot about host–pathogen interaction and adaptation of organisms to conditions of life. Additionally, insect‐derived antibacterial and antifungal peptides and proteins are considered for their potential to be applied as alternatives to antibiotics. While Drosophila melanogaster is used to study the genetic aspect of insect immunity, Galleria mellonella serves as a good model for biochemical research. Given the size of the insect, it is possible to obtain easily hemolymph and other tissues as a source of many immune‐relevant polypeptides. This review article summarizes our knowledge concerning G. mellonella immunity. The best‐characterized immune‐related proteins and peptides are recalled and their short characteristic is given. Some other proteins identified at the mRNA level are also mentioned. The infectious routes used by Galleria natural pathogens such as Bacillus thuringiensis and Beauveria bassiana are also described in the context of host–pathogen interaction. Finally, the plasticity of G. mellonella immune response influenced by abiotic and biotic factors is described.     

The ultrastructure and ultracytochemistry of the basement membrane of the Galleria mellonella fat body

Summary The fat body lobes of Galleria mellonella are surrounded by basement membrane — a fine granular layer of connective tissue. This membrane has an affinity for ruthenium red. The results obtained after treatment of the fat body with neuraminidase, hyaluronidase, phospholipase C and proteolytic enzymes suggest that glycoproteins and phospholipoproteins are constituents of this basement membrane. The basement membrane also has the ability to bind concanavalin A-peroxidase, which is associated with the presence of mannoside residues.The preliminary results of these studies were presented at the IX Conference of Electron Microscopy, Gdask, Poland (Dutkowski, 1975)I am greatly indebted to Professor A. Przececka for her encouragement to undertake this study. The excellent technical assistance of Mrs. K. Mroziska, Mrs. Z. Kamiska and the engineering staff of the Laboratory is gratefully acknowledged     

A new cell line from larval fat bodies of the bollworm, Helicoverpa armigera (Lepidoptera: Noctuidae)

Summary A new cell line, designated IOZCAS-Ha-I, was initiated from the fat body of larvae of Helicoverpa armigera (Lepidoptera: Noctuidae) in TNM-FH medium containing 10% fetal bovine serum. Spherical cells were predominant among the various cell types. The cell line showed a typical lepidopteran chromosome pattern ranging from 58 to 239 chromosomes in the majority of the cells, it was confirmed to have originated from the H. armigera by the DNA amplification-fingerprinting polymerase chain reaction (DAF-PCR) technique. The new cell line was only slightly susceptible to the multiple nucleocapsid nuclear polyhedrosis viruses (NPV) from H. armigera.     

Receptor-mediated endocytosis of lipid and lipophorin by the larval fat body, adult ovary and testis in the wax moth Galleria mellonella

Lipophorin (Lp) in the hemolymph of insects is known to selectively deliver lipids from sites of absorption or synthesis to sites of storage and utilization, such as the fat body, ovary and testis; however, no study regarding this has been reported in Galleria mellonella. In the present study, we examined the process by which Lp is taken up into the larval fat body, adult ovary and adult testis, and the transfer of lipid by Lp to these tissues in Galleria mellonella. To investigate the involvement of a receptor in Lp endocytosis, the larval fat body, adult ovary and adult testis were incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)‐Lp, FITC‐Lp plus unlabeled Lp, and FITC‐Lp plus suramin, a receptor endocytic inhibitor. The amounts of FITC‐Lp in the three tissues were significantly decreased in the presence of unlabeled Lp and suramin, indicating that endocytosis of Lp by the tissues is mediated by a receptor. To examine the transfer of lipid by Lp, the tissues were incubated for 1 h at room temperature with 1,1′‐dilinoleyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate (DiI)‐Lp, DiI‐Lp plus unlabeled Lp, and DiI‐Lp plus suramin. The transfer of lipid by Lp was inhibited in the presence of unlabeled Lp and suramin, which is consistent with a receptor‐mediated process. Our results show that the transfer process of lipid by Lp and uptake of Lp itself is by receptor‐mediated endocytosis.     

Electron microscopical-immunocytochemical evidence of ecdysteroids in the prothoracic gland of Galleria mellonella

Summary Fixation of prothoracic glands of Galleria mellonella with a solution containing saponin permits immunocytochemical staining of the entire gland. By this means ecdysteroids were demonstrated electron microscopically to be present in the hyaloplasm and microtubules.Supported by Sächsische Akademie der Wissenschaften zu Leipzig     

Correlation between Gliotoxin Production and Virulence of Aspergillus fumigatus in Galleria mellonella

Thanatephorus cucumeris is a ubiquitous fungus responsible for many types of plant diseases worldwide. All isolates from infected Hevea brasiliensis trees secreted pectolytic enzymes; polygalacturonase (PG), pectin lyase (PL) and cellulolytic enzymes; beta-glucosidase and cellobiase in culture. The extracts of the rubber tree leaf tissues, inoculated with T. cucumeris did not show any PG activity. However, PL activity was detected in tissue with the establishment of the infection. The levels of beta-glucosidase, an inherent enzyme in Hevea spp. increased rapidly following infection. However, cellobiase was detected only with the initiation of infection. Molecular weights of PG in all isolates were similar and in the range of 53,000 to 58,000. PL also followed the same pattern showing a molecular weight around 39,000.     

Primary structure of apolipophorin-III from the greater wax moth,Galleria mellonella

The complete amino acid sequence of apolipophorin-III (apoLp-III), a lipid-binding hemolymph protein from the greater wax moth,Galleria mellonella, was determined by protein sequencing. The mature protein consists of 163 amino acid residues forming a protein of 18,075.5 Da. Its sequence is similar to apoLp-III from other Lepidopteran species, but remarkably different from the apoLp-IIIs of insects from other orders. As shown by mass spectrometric analysis, the protein carries no modifications. Thus, all of its known physiological functions, including its recently discovered immune response-stimulating activity, must reside in the protein itself.     

Toxicity of mycotoxins of Fusarium sambucinum for feeding in Galleria mellonella

Fusarium sambucinum Fuckel showed antifeedant activity towards larvae of Galleria mellonella L. when incorporated into insect diet. The activity appeared mostly due to the concentration of trichothecenes present in the fungal extracts. Diacetoxyscirpenol and neosolaniol showed similar levels of activity and were significant potent antifeedants against larvae at 50 and 100 ppm. On the contrary, enniatin B showed no activity up to 100 ppm.     

Cloning and expression of male-specific protein (MSP) from the hemolymph of greater wax moth, Galleria mellonella L

Male-specific protein (MSP) is a soluble protein that accumulates in high amounts in the hemolymph and other organs of adult male wax moth. The MSP was purified from adult male wax moth by gel filtration and reversed phase column chromatography, and its amino acid sequence was determined. Because of blocked N-terminus, several internal amino acid sequences of MSP were obtained by the in-gel digestion method using trypsin. RT-PCR was conducted using degenerate primers designed from the internal amino acid sequences. 5'-RACE PCR was used to obtain the complete coding region and 5'-UTR sequence. The full length MSP cDNA sequence encodes a 239 amino acid polypeptide with an 18 amino acid signal peptide. The putative mature MSP has a molecular mass of 24,317 Da and an isoelectric point (pI) of 6.00, but shows a molecular mass of 27 kDa on SDS-PAGE. Sequence alignment showed a significant similarity between MSP and juvenile hormone binding proteins (JHBPs) of several lepidopteran species, including G. mellonella.     

Influence of diet upon the composition of pheromone volatiles in Galleria mellonella (greater wax moth) males

The composition of pheromone volatiles from calling males of the greater wax moth Galleria mellonella reared on their native (ND) or artificial (AD) diet was studied by GC-MS. The comparison of effluvium of two laboratory races of insects fed ND or AD was carried out for populations of greater wax moth from three regions of Russia. The experiments suggest that the AD changes the ratio and amounts of major components of pheromone, which are different for different regions. Arch. Insect Biochem. Physiol. 36:129–138, 1997. © 1997 Wiley-Liss, Inc.     

Susceptibility of Larvae of Galleria mellonella to Infection by Aspergillus fumigatus is Dependent upon Stage of Conidial Germination

The ability of conidia of the human pathogenic fungus Aspergillus fumigatus to kill larvae of the insect Galleria mellonella was investigated. Conidia at different stages of the germination process displayed variations in their virulence as measured using the Galleria infection model. Non-germinating (‘resting’) conidia were avirulent except when an inoculation density of 1 × 10⁷ conidia per insect was used. Conidia that had been induced to commence the germination process by pre-culturing in growth medium for 3 h were capable of killing larvae at densities of 1 × 10⁶ and 1 × 10⁷ per insect. An inoculation density of 1 × 10⁵ conidia per insect remained avirulent. Conidia in the outgrowth phase of germination (characterised as the formation of a germ tube) were the most virulent and were capable of killing 100% of larvae after 5 or 24 h when 1 × 10⁷ or 1 × 10⁶ conidia, that had been allowed to germinate for 24 h, were used. Examination of the response of insect haemocytes to conidia at different stages of the germination process established that haemocytes could engulf non-germinating conidia and those in the early stages of the germination process but that conidia, which had reached the outgrowth stages of germination were not phagocytosed. The results presented here indicate that haemocytes of G. mellonella are capable of phagocytosing A. fumigatus conidia less than 3.0 μm in diameter but that conidia greater than this are too large to be engulfed. The virulence of A. fumigatus in G. mellonella larvae can be ascertained within 60–90 h if infection densities of 1 × 10⁶ or 1 × 10⁷ activated conidia (pre-incubated for 2–3 h) per insect are employed.     

Humoral immune response upon mild heat-shock conditions in Galleria mellonella larvae

Larvae of Galleria mellonella exposed to mild heat-shock (38 degrees C) showed an enhanced humoral immune response after microbial infection in comparison to infected animals grown at 28 degrees C. This enhanced response was manifested by increased expression of antimicrobial peptide (AMP) genes leading to enhanced antimicrobial activity in the hemolymph. We found an increased level of Hsp90 and changes in the level of a 55kDa protein recognized by anti-Hsp90 antibodies in fat bodies of infected animals reared at 28 degrees C as well as in uninfected animals exposed to elevated temperature. Pre-treatment of animals with an inhibitor of Hsp90, 17-DMAG, prior to immunization resulted in increased expression of AMP genes encoding gallerimycin and cecropin at 38 degrees C. This observation was correlated with the changes in Hsp90 protein and increased level of 55kDa protein. Also G. mellonella larvae pre-treated with 17-DMAG and exposed to mild heat-shock for 30min showed an increased survival rate after infection with entomopathogenic bacteria Pseudomonas aeruginosa. We also show the effect of 17-DMAG on the phosphorylation state of ERK MAP kinase. We postulate that Hsp90 may play a significant role in converging pathways involved in the insect immune response and heat-shock.     

Immunocompetence of Galleria mellonella: sex- and stage-specific differences and the physiological cost of mounting an immune response during metamorphosis

The antibacterial immune response of the wax moth, Galleria mellonella, was analysed by use of an inhibition zone plate assay. We demonstrated significant stage-specific differences as the immune response was most effective in the pupal, next the larval and then the adult stage. In addition, we demonstrated that an immune challenge at the onset of, or during metamorphosis does not increase nor decrease the strength of the antibacterial immune response in the subsequent developmental stage(s). These findings illustrate that induced immunity is not preserved during metamorphosis but also deny any cost to the immune system itself. However, an immune challenge does induce a significant shortening of the direct development time and affects the mass loss during metamorphosis in a sex-dependent manner: males emerged smaller whereas the mass of females was not significantly affected. These observations indicate that there are sex-specific costs to mounting an immune response during metamorphosis which affect physiological traits, implicating a trade-off between immunity and development.